The Mechanism Study Of Testosterone Protecting PC12 From Apoptosis Induced By Aβ_25-35

Alzheimer's disease is a popular degenerative disease of nervous system among the elder. The pathogenesis of it has not been figured out completely. Currently, it is considered that the decrease of sex hormone following aging is one of the essential etiological factors resulting in AD. It will be of great importance to illustrate further about the role sex hormone in vivo playing in the occurence of AD for the study of its pathogenesis and its clinical prevention and treatment.This task clarifies the testosterone's effect on the apoptosis of PC12 in vitro, providing some theoretical basis for the further study of AD pathogenesis and its clinical treatment.This task comprises of three parts below.Part I:The effect of testosterone on the PC 12 cells activity induced by Aβ25-35Adopt MTT analysis to detect the effect of testosterone on AP25-35 induced PC 12 apoptotic activity. Incubating PC 12 for 24 hours in culture solution of different final concentration beforehand, PC 12 activity decrease tremendously and the decrease degree is concerned with AP25-35 concentration. Its IC50 is about 20μM. Next we protect it in testosterone culture solution of different concentration for 12 hours. Thirdly, induce PC 12 to apoptosis with 20μM Aβ25-35. It is proved that the decrease of PC 12 activity is inhibited obviously and it is increased with the concentration of testosterone growing. Its IC50 is about 100μM. Add 100μM flutamide into the testosterone culture solution. Then induce PC 12 apoptosis with 20μM Aβ25-35. The protection of testosterone to PC 12 is clearly inhibited. Judging from MTT, testosterone can inhibit PC 12 apoptosis induced by Aβ25-35 significantly, which may affect through androgen-receptor pathway.PartⅡ:The morphology study of testosterone effect on PC 12 apoptosis induced by Aβ25-35.Adopt Hochest-PI two tone dyeing to investigate the effect of testosterone on PC 12 apoptosis induced by Aβ25-35. In this study, we set the four treatment groups:Control group,Pre-protection group of testosterone,Aβ25-35 treatment group,Flutamide treatment group. Hochest-PI double staining revealed that the number of apoptotic PC 12 cells increased significantly in Aβ25-35 treatment group,while decreased clearly in the testosterone pre-protection group.It also revealed that the effect of testosterone was inhibited obviously if pre-intervention with flutamide. It is morphologically proved the inhibition effect of testosterone exerting on PC 12 cell apoptosis.PartⅢ:The effect of testosterone on Bcl-xl,Bax expression in apoptosis PC 12 induced by AP25.35.The Exp 3 and the Exp 4 are included in this part. Each experiment contains four treatment groups:Control group,Pre-protection group of testosterone,AP25-35 treatment group,Flutamide treatment group. Western-blot Immunoblotting was adopted to detect the expression of protein Bax,Bcl-xl in the exp 3. We detected the expression of Bax,Bcl-xl mRNA by adopting Real-time PCR in the Exp 4. Experimental results show that the expression of Bax protein and Bax mRNA were significantly up-regulated, Bcl-xl protein and Bcl-xl mRNA expression was significantly lower (P<0.05) in Aβ25-35 treatment group;Protection PC 12 cells after testosterone,the expression of Bax protein and Bax mRNA were significantly decreased, Bcl-xl protein and Bcl-xl mRNA expression was significantly increased (P<0.01); After the intervention of flutamide,the expression of Bcl-xl protein and Bcl-xl mRNA was significantly decreased, while Bax protein and Bax mRNA was significantly increased compared with the protection of Testosterone (P<0.05). So with the above results we conclude that anti-apoptotic effect of testosterone may be associated with increasing Bcl-xl, reducing the expression of Bax, and its effect of anti-apoptosis role may be mediated through the androgen receptor.In summation, the conclusions we concluded are as follows.Aβ25-35 can induce apoptosis of PC 12 cell significantly in vitro;2) Testosterone can inhibit apoptosis of PC 12 induced by Aβ25-35 and the effect may work through androgen-receptor pathway. 3) The potential mechanism of anti-apoptosis role of testosterone on PC 12 may be achieved through inhibiting Bax and up-regulating Bcl-xl expression at the levels of gene transcription and protein expression.